Measles virus normally causes an acute respiratory infection followed by patient recovery, except in underdeveloped countries where infections lead to a significant mortality. On rare occasions, measles virus causes a prolonged fatal neurological disease, subacute sclerosing panencephalitis (SSPE), where the genome of the recovered SSPE virus has significant alterations, compared to the parental measles virus. The overall goals of this proposal are to study the mechanism of the two forms of editing which alter viral RNAs and the mechanism of measles RNA replication. P mRNA editing involves the incorporation of a nontemplated G nucleotide at a specific sequence, yielding a new P-like mRNA encoding the nonstructural V protein. The second type of editing, biased hypermutation, results in non- random U to C changes which are characteristic of SSPE virus isolates. We will characterize the P mRNA editing reaction in vitro and determine if the host cell has an effect on editing during measles virus or SSPE virus infection of neural and non-neural cell lines. We will test if biased hypermutation occurs during measles virus infection of any of these cells. An in vitro assay for viral biased hypermutation is being developed to test the roles of the virus polymerase and/or the host cell-specifically the cellular double stranded RNA unwinding RNA modifying enzyme. Infectious defective interfering (DI) cDNAs will be prepared to study both measles virus transcription and editing as well as RNA replication. A cDNA representing an internal deletion DI containing the N gene and a truncated P gene will be utilized for the editing studies and a cDNA of a known copy- back DI RNA will be used for replication studies. The cDNAs will be cloned downstream of the T7 RNA polymerase promoter in a plasmid and transfected into cells that have been co-infected with a recombinant vaccinia virus expressing T7 RNA polymerase to provide for the synthesis of DI RNA and wild-type measles virus as the helper virus. The transcription/editing template will be used to study the effect of the wild-type measles or SSPE virus RNA polymerases on editing in vivo and to identify the required cis RNA sequences by site-directed mutagenesis. The copy-back DI cDNA will be used to determine the RNA sequences required for replication and packaging of viral RNA as well as biased hypermutation. These approaches should yield a better understanding of viral RNA synthesis as well as ultimately allow for the study of other aspects of measles virus transcription, mRNA processing and protein function.